Lipoxygenase enzymes belong to a large, multigene-family involved in the regulation and biosynthesis of a number of biologically active compounds. They have been implicated in a number of processes including senescence, plant growth and development, mobilization of lipid reserves during seed germination disease resistance responses, vegetative storage proteins and in the production of flavor and scent compounds. The present invention is directed to the involvement of lipoxygenase enzymes in the production of flavor compounds or precursors and is particularly directed to the role of lipoxygenase enzymes in the fruit of Vitis vinifera (grape).
There is evidence in the literature of a connection between lipoxygenase enzyme (LOX) activity and the production of compounds associated with flavor in grape. See e.g.,Cayrel et al., Amer. J. of Ecology and Viticulture 34:77-82 (1983); Crouzet et al., Progress in Flavour Research 1984, Proceedings of the 4th Weurman Flavour Research Symposium (J. Adda ed.) Elsivier Science Publishers, (1985); Waldman and Schreier, J. of Agri. Food Chem., 43:626-630 (1995); O""Conner and O""Brien, Food Enzymology 1: 337-372 (1991); Gardner, HW in: Flavor Chemistry of Lipid Foods. Eds. Min, D. B. and Smouse, T. H. The American Oil Chemists"" Society (1989); Angerosa, F., et al. J. of Agri. Food Chem. 47: 836-839 (1999); and Hanataka, Food Review International, 12:303-350 (1996). Oxidation of linoleic and linolenic acids by LOX produces C9 and C13 hydroperoxides that can be further modified by other enzymes to produce C6, C9, and C12 compounds with characteristic flavors and aroma. Such C6 compounds associated with flavors and aroma include 3Z-hexenal, 3E-hexenal, 2E-hexenal, 3Z-hexenol, 3E-hexenol, 2E-hexenol, n-hexanal and n-hexenol. Such C9 compounds associated with flavors and aroma including 3Zxe2x95x906Z-nonadienal, 2E-6Z-nonadienal, 3Z-6Z-nonadienol, 2E-6Z-nonadienol, 9-oxo-nonanoic acid, 3Z-nonenal, 2E-nonenal, 3Z-nonenol, 2E-nonenol and C12 flavor and aroma compounds include 12-oxo-9Z-dodecenoic acid and 12-ox-10E-dodecenoic acid.
Production of these volatiles depends on the initial fatty acid substrate, the particular LOX isozyme, and the presence of other enzymes required for formation of the different volatile compounds. In addition, free radicals released during the oxidation of fatty acids by LOX can potentially induce cooxidation of carotenoid compounds yielding a number of flavor and aroma compounds.
Lipoxygenase has been characterized in a number of species and in most cases is encoded by a member of a large gene family. In soybean, LOX is comprised of a highly conserved multigene family consisting of at least eight members. There are at least five vegetative LOX (VLX) genes involved in nitrogen storage function, that represent a major storage protein in soybean leaves. Of these, VLXD proteins increase in sink limited soybeans. VLXC+D are degraded preferentially during pod maturation, and VLXC has a dual role as a storage protein and a cytosolic enzyme. There are also three LOX genes found in the seeds, LX 1, LX2 and LX3. Analysis of cloned LOX genes reveals a highly conserved 70 kd globular domain and a 30 kd beta-barrel domain. In addition it has been observed that the N-terminus of the LOX genes is extremely divergent. (Howard Grimes presentation at the 1999 American Society of Plant Physiology titled, xe2x80x9cLipoxygenase Function in Assimilate Partitioningxe2x80x9d) In most plants analyzed, there exist LOX genes with similarity to both the vegetative and seed type lipoxygenases found in soybean.
Despite the identification and cloning of LOX genes in a number of plant species including soybean (Glycine max.), Solanum tuberosum, Hordeum vulgare, Oryza sativa, Arabidopsis thaliana, Cucumis sativa and Prunus dulcis, LOX genes have not been cloned from Vitis vinifera and there exists no source to obtain pure lipoxygenase from grape. Accordingly, there exists a desire in the art for pure Vitis vinifera LOX.
The present invention provides purified and isolated polynucleotide encoding a Vitis vinifera LOX polypeptide selected from the group consisting of: a) a polynucleotide comprising the sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4, b) a DNA which hybridizes under moderately stringent conditions to the non-coding strand of the polynucleotide of (a); and c) a DNA which would hybridize to the non-coding strand of the polynucleotide of (a) but for the redundancy of the genetic code. The polynucleotide of the invention is preferably a DNA molecule and is more preferably a cDNA molecule. Alternatively, the DNA is a wholly or partially chemically synthesized DNA molecule. According to another embodiment of the invention the polynucleotide is an a) anti-sense polynucleotide which specifically hybridizes with the polynucleotide as set forth in SEQ ID NO: 2 or SEQ ID NO: 4 b) a DNA which hybridizes under moderately stringent conditions to the non-coding strand of the polynucleotide of (a); and c) a DNA which would hybridize to the non-coding strand of the polynucleotide of (a) but for the redundancy of the genetic code. The invention also provides polynucleotides where the LOX encoding sequence is operably linked to a heterologous promoter. The invention also provides expression constructs, comprising the polynucleotide of the invention, as well as host cells transformed or transfected with a polynucleotide or expression construct of the invention. The invention also provides polynucleotides of the invention operably linked to a heterologous promoter, and host cell polynucleotides operably linked to a heterologous promoter.
Host cells transformed or transfected according to the invention include those which are Vitis vinifera cells. The invention also provides transformed plants comprising host cells transformed or transfected with the LOX gene. Transformed plants of the invention include those wherein the expression construct comprises a polynucleotide encoding a Vitis vinifera LOX polypeptide operably linked to a heterologous promoter. According to one preferred embodiment the transformed plant is Vitis vinifera. Alternatively, transformed cells include microorganisms including those active in fermentation reactions and including those selected from the group consisting of yeast and bacteria.
The invention also provides a purified and isolated Vitis vinifera LOX polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Amino acid SEQ ID NOS: 1 and 3 were determined by analysis of polynucleotide SEQ ID NOS: 2 and 4, respectively, and comparison with known LOX amino acid sequences. The availability of purified LOX enzyme provided by the invention makes possible the use of the enzyme to modify food flavors by contacting a food or other comestible with a quantity of purified and isolated Vitis vinifera LOX polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 under conditions selected to modify the flavor characteristics of the comestible. Food products, which can be treated according to the invention, include comestibles that are beverages. It is contemplated that the invention will be particularly useful in the treatment of beverages that are fermentation products and most preferably wine.
In particular, the invention provides a method for modifying the flavor of a fermented beverage, including wine, comprising the step of fermenting said beverage in the presence of a microorganism transformed or transfected with an expression construct comprising a LOX polynucleotide according to the invention. Preferred microorganisms include those selected from the group consisting of yeast and bacteria.
Cloning of the Vitis gene encoding lipoxygenase enables the heterologous production of pure lipoxyegenase enzyme in a protein expression vector. Production of sufficient quantities of enzyme allows analysis of its effect on flavor production in wine and grape juice. In addition, the cloned gene enables study of the native level of gene expression in response to environmental or viticultural influences. Further, the cloned gene can be used to produce transgenic plants to modify the level of gene expression to produce optimal levels of lipoxygenase in the grape.